Membrane based detection of genetically modified organisms in some representatives food

Recently, DNA-based techniques became very common for the detection of genetically modified organisms (GMOs) in food products. For rapid and easy detection of GMOs, polymerase chain reaction (PCR) screening methods, which amplify common transgenic elements, are applied in routine analysis. Incorporation of PCR and membrane method introduced in this study offer an alternative detection of GMOs. In this study, a total of 32 samples and three certified reference materials were tested for the existence of the 35S promoter of cauliflower mosaic virus (CaMV) and 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) gene residues. Dot blot screening system introduced in this study can be routinely used as a semi-quantitative screening of GMOs.     

Monitoring of Roundup™ Ready soybean in Guangdong province in China

This study was aimed at monitoring the presence of Roundup™ Ready soybean (RRS) in the market (660 samples) and fields (630 samples) in Guangdong Province in China using polymerase chain reaction (PCR) method. According to the gene sequence transgened to Roundup Ready™ soybean, four primer pairs, aiming at cp4-epsps, lectin, CaMV 35S promoter and nos terminator were designed and PCR method to detect the GM soybean has been established. The detection results demonstrate the presence of one Roundup™ Ready soybean seed sample from 60 samples in Shenzhen City. No soybean plant samples from Guangdong Province were found.     

A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean,maize and rice in highly processed products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of the GMOs are necessary in order to verify compliance with labeling requirements. There are few effective screening methods for highly processed GM (genetically modified) products. Four genes (CP4-EPSPS, Cry1A(b), BAR, and, PAT) are common exogenous genes used in commercialized transgenic soybean, maize, and rice. In the present study, a multiplex nested polymerase chain reaction (PCR) method was developed to simultaneously detect the four exogenous genes and one endogenous gene in two runs. We tested eleven representative highly processed products samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, soybean refine oil, soybean salad oil, maize oil, maize protein powder, maize starch, maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products except for soybean refined oil, soybean salad oil and maize oil, and the sensitivity was 0.005%. These results indicate that multiplex nested PCR is appropriate for qualitative detection of transgenic soybean, maize and rice in highly processed products except for refined oil.     

Multiplex PCR system to track authorized and unauthorized genetically modified soybean events in food and feed

A diverse range of genetic elements has been used to develop genetically modified organisms (GMOs) over the last 18 years. Screening methods that target few elements, such as the Cauliflower Mosaic Virus 35S promoter (P-35S) and Agrobacterium tumefaciens nopaline terminator (T-nos), are not sufficient to screen GMOs. In the present study, a multiplex PCR system for all globally commercialized GM soybean events was developed to easily trace the events. For this purpose, screening elements of 24 GM soybean events were investigated and 9 screening targets were selected and divided into three individual triplex PCR systems: P-35S, ribulose-1,5-bisphosphate carboxylase small subunit promoter of Arabidopsis thaliana, T-nos, T-35S, pea E9 terminator, open reading frame 23 terminator of A. tumefaciens, proteinase inhibitor II terminator of potato, acetohydroxy acid synthase large subunit terminator of A. thaliana, and the revealed 3′ flanking sequences of DP-305423-1. The specificity of the assays was confirmed using thirteen GM soybean events as the respective positive/negative controls. The limit of detection of each multiplex set, as determined using certified reference materials of specific GM events, ranged from 0.03 to 0.5%, depending upon target. Furthermore, 26 food samples that contained soybean ingredients, which were purchased from the USA, China, Japan, and Korea, were analyzed, 17 of which contained one or more GM soybean events. These results suggest that the developed screening method can be used to efficiently track and identify 24 GM soybean events in food and feed.     

Detection of Roundup Ready soy in highly processed products by triplex nested PCR

Till now, there still has not an effective detection method for the highly processed GM (genetically modified) products. A novel method of the triplex nested PCR, was developed for the sensitive detection of several foreign genes (Lectin, CaMV 35S, CTP, CP4-EPSPS, NOS) in highly processed products. We detected seven representative highly processed products (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, crude soybean oil, soybean refine oil, soybean salad oil) by the triplex nested PCR. The first triplex PCR cannot detect the insert signals in the processed products, and the sensitivity is 0.5%. However the second triplex PCR, which can simultaneously detect RR soybean targets with a sensitivity of 0.005% in the triplex nested PCR. The result indicates the advanced level of the method for the GM products detection. It is a flexible assay to detect the RR soybean in highly processed products.     

Quantitative competitive PCR for the detection of genetically modified organisms in food

Present polymerase chain reaction (PCR) detection methods only allow the qualitative detection of GMO in food without quantitation of the GMO content. Clearly, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. This quantitative competitive (QC) PCR was first described in the early nineties and is widely used nowadays.

We have developed and evaluated QC–PCR systems for the quantitative detection of Roundup Ready^TM soybean (RRS) and Maximizer maize (MM) in food samples. Three DNA fragments differing from the GMO specific sequences by DNA insertions were constructed and used as internal standards in QC–PCR. These standards were calibrated by co-amplifying with mixtures containing defined amounts of RRS DNA and MM DNA, respectively. The calibrated QC–PCR systems were applied to several commercial food samples containing RRS and to three certified RRS flour mixtures (Fluka standards). Recently, quantitative methods for the detection of RRS were successfully tested in a collaborative study involving twelve European control laboratories. Thus, QC–PCR methods will allow to survey “de minimis thresholds” of GMOs in food.     

Results of an interlaboratory assessment of a screening method of genetically modified organisms in soy beans and maize

The preliminary results on an interlaboratory trial on the detection of genetically modified organisms (GMO) are presented. The method applied is based on the detection of modified DNA using the polymerase chain reaction (PCR) for amplification. The amplified fragments analysed are derived from the 35S promotor and the NOS terminator used for modification and are present in 26 from the 28 GMOs currently already approved or under approval by the competent authorities. This method fits as a screening method indicating the presence of GMO in food. However, it does not allow an identification of the kind of GMO present in the samples. Samples of soybean and maize flour containing 0%, 0.1%, 0.5% and 2% GMO had been prepared for this study and are also already commercially available. In this paper the combined results from 27 laboratories are presented, indicating that on average the probability of false positive or false negative results is only about 1% for soybeans and below 5% for maize.     

Disposable genosensor, a new tool for the detection of NOS-terminator, a genetic element present in GMOs

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. An indicator based electrochemical disposable genosensor for the voltammetric detection of NOS-terminator, a genetic element present in GMOs is described as a possible substitute method for the common technique of gel electrophoresis and fluorescent image analysis. The biosensor relies on the immobilization of the 25-mer single stranded oligonucleotides (probe) related to NOS-terminator DNA sequence and the relative binding of this sequence with the polymerase chain reaction (PCR) amplified samples from certified reference material (CRM) of Roundup Ready soybean (Fluka) at a screen printed electrode (SPE). The extent of hybridization between the probe and target DNA is determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB), as the hybridization indicator. The difference between the MB signals, obtained from the hybrid modified and probe modified SPEs, is used to detect GMOs from PCR amplified DNA samples. Numerous factors affecting the hybridization and indicator binding reactions are optimized to maximize the sensitivity.     

Comparison of simplex and duplex real-time PCR for the quantification of GMO in maize and soybean

This paper focuses on the determination of the GMO content of maize and soybean samples using real-time PCR, comparing simplex and duplex PCR. The total DNA content of samples was determined by amplifying part of a maize gene encoding a lipid transfer protein, or part of a soybean lectin gene. The transgenic DNA was quantified by amplifying part of the CaMV 35S promoter. The importance of preparation and conservation of standards as well as the relevance of DNA extraction protocol on the variability of results are discussed. For the determination of low GMO content, limitation in the number of copies of the target gene to be amplified is considered. For samples with a theoretical GMO content of 1%, corresponding to the legal threshold for labelling, the value determined by duplex real-time PCR ranged from 0.85% to 1.20%. Both real-time simplex and duplex PCRs allowed identification of GMO free samples without ambiguity.     

Event-specific qualitative and quantitative PCR detection of LY038 maize in mixed samples

With the development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative PCR detection methods have become the internationally agreed state-of-art. This paper describes the characterization and event-specific quantitative detection of LY038 maize insert with the application of reference molecule. The flanking regions were characterized by Inverse-PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right flanking sequence. In the qualitative PCR assay, two PCR systems were established with the event-specific and specie-specific primers respectively, and the limit of detection (LOD) was 0.1% (approximates to 37 haploid genome copies). In the quantitative TaqMan real-time PCR assay, a reference molecule was constructed by recombinant PCR and standard curves were set up. By using the reference molecule, we obtained standard curves with good linearity and relatively high efficiency of PCR reaction. The results indicated the usability of the plasmid as standard material. From above results, we believed that the established event-specific qualitative and quantitative PCR systems for LY038 maize in this study were acceptable and suitable for LY038 maize detection in mixed samples.     

An event-specific qualitative and real-time PCR detection of 98140 maize in mixed samples

The purpose of this study is to establish a reliable detection method specific for event 98140, a type of multiple herbicide-resistant corn. Based on the 3′- flanking sequence of event 98140, qualitative and quantitative PCR detection assays were developed. The results revealed the LOD of 0.05% of GM maize for qualitative PCR assay and 4 transgenic haploid genome copies for quantitative PCR assay used in this study. The LOQ was 20 transgenic haploid genome copies, and based on this, as low as 0.05% of 98140 genomic DNA could be accurately and quantitatively detected by means of the quantitative method. Two mixed corn samples, with known 98140 contents (2% and 0.5%), were used to verify the developed real-time PCR system, and the expected results were observed. The results indicated that the developed event-specific PCR methods could be used for the identification and quantification of maize line 98140.     

A general multiplex-PCR assay for the general detection of genetically modified soya and maize

The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for the detection of GMOs are necessary in order to verify compliance with labelling requirements. In the past few years, different PCR-based methods for the specific detection of the most economically important GMOs have been proposed. A molecular screening method based on multiplex-PCR that involves amplification of specific soya or maize sequences from plant DNA and the amplification of 35S promoter and NOS terminator for the detection of genetically modified soya and maize was developed. The m-PCR assay discriminated the GMO very quickly, reproducibly and in a cost saving and less time-consuming way. It is a flexible assay to conduce a preliminary GMO screening for detection of genetically modified soya and maize.     

Specific,semi-quantitative detection of the soybean allergen Gly m Bd 30K DNA by PCR

A semi-quantitative PCR-based system has been developed for detection of DNA sequences for the soybean allergen Gly m Bd 30K. The selected primers were highly specific for soybean and did not show amplification from a panel of legume relatives. Repeatability was assessed in a spiking experiment of soybean in wheat flour (0.0001–100% soybean), using known standards for comparison of the amount of output DNA from different PCR reactions. The frequency of PCR reactions with successful amplification of the soybean allergen sequence was highly dependent on initial target DNA concentration, and showed a rapid sigmoidal decrease, when target concentration approached the detection limit (0.01%). For samples with successful amplification there was a good correlation between the initial amount of soybean in the mixture and the output from PCR, when suitable block designs were used to control experimental errors. The simple approach used for quantification in this study proved efficient for assessment of homogeneity of self-prepared soybean bars used for provocation tests of food allergic patients in clinical practice. In addition the method was efficient in detecting soybean allergen sequences in a number of processed foods.     

Transboundary movement of genetically modified organisms in India: Current scenario and a decision support system

Genetically modified (GM) crops have benefited global agriculture by introduction of superior traits for better agronomic performance, ensuring nutritional security and mitigating climate change. In India, to meet the demand of burgeoning population and to withstand the changing climate, GM crops would play an important role. Since 1997, GM crops are being imported through Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, the designated nodal organization for quarantine processing and import of GMOs (referred to GM planting material in present context) for research purposes. In the present study, an attempt has been made to analyze the trend of import of GMOs. Till the end of 2015, 205 consignments of fifteen GM crops have been imported from 19 countries by public and private sector. Detailed analysis of diversity in traits of imported GM events and imported stacked traits in cotton and maize has been made. In the recent past, four consignments of GMOs have been exported for research purposes. Involvement of public/private sector in transboundary movement of GMOs was evaluated. Along with quarantine processing of imported/exported GMOs, molecular testing for specific transgenic elements as claimed by the importer/exporter is also carried out employing polymerase chain reaction (PCR) and real-time PCR based markers. Efficient detection strategies based on GMO matrix as a decision support system, loop-mediated isothermal amplification and multi-target real-time PCR-based systems have been developed. The data presented herein would provide a decision support system to check for authorized/unauthorized GMOs in food and supply chain.     

Screening new gene markers for gluten detection in foods

In the present work, three DNA sequences encoding wheat proteins (α2-gliadin, agglutinin isolectin and thioredoxin h) were compared to trace gluten-containing cereals in food products. Quantitative real-time PCR methods using hydrolysis probes were successfully developed to target the three sequences for the detection of wheat DNA. The comparison of the three systems highlights the best sensitivity when tracing the α2-gliadin marker sequence, showing an absolute limit of detection (LOD) of 2 pg of wheat DNA and a relative LOD of 0.005% (50 mg/kg) of wheat in soybean, which corresponds to 4.5 mg/kg of gluten. All the systems reveal high specificity for detecting other gluten-containing cereals, such as barley and rye. Therefore, the developed real-time PCR systems can be used as non-immunological tools to confirm the presence of gluten-containing cereals in foods, towards the safety of celiac patients and wheat allergic individuals.     

Applicability of a rapid method based on immunomagnetic capture-fluorescent PCR assay for Campylobacter jejuni

In order to develop a rapid method which can check Campylobacter jejuni in foods and water effectively, an immunomagnetic capture-fluorescent PCR (IMC-FPCR) method was established in this paper. The reported method involves isolation of the target pathogen by immunocapture prior to the fluorescent PCR step, therefore the immunomagnetic-beads for Campylobacter were developed, and two groups of primer/probe, which targeted for the species special sequence of flaA gene and hipO gene for C. jejuni were designed. Result indicated that the method permits direct detection of the pathogen without an enrichment step and can be performed in approximately 8 h. The assay results are positive for all of the isolates of C. jejuni with a detection limit of approximately 10 cfu/ml, are negative for Campylobacter coli and several other bacteria. During the experiment of 300 samples of foods, four of them were positive and the result was the same as traditional biochemical identification. Application of the method to 100 samples of water, one of them was positive but the result was negative with traditional method. Thus, the IMC-FPCR assay provide not only a rapid, sensitive method for quantitative detection of C. jejuni, but also a potential method for detecting of C. jejuni of viable but non-culturable (VBNC) state.     

Development and application of DNA analytical methods for the detection of GMOs in food

The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products.

Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil.

If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker.

Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity.

Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers.     

Authentication of Edible Bird's nests by TaqMan-based real-time PCR

Edible Bird's nests (EBN) have been adulterated with less expensive materials, including white fungus, agar–agar, fried pigskin, egg white and red seaweed, for several years. To protect consumers and regulate the EBN market, it is necessary to establish a robust method for detecting these adulterants in EBN. Herein, we established a TaqMan-based real-time polymerase chain reaction (PCR) assay to specifically detect EBN component and four common adulterants: white fungus, agar, pigskin and egg white. Therefore, five sets of primers and probes were designed for these five components. The assays were specific and reproducible, and the relative detection limits were 0.5% EBN in white fungus, 0.001% white fungus in EBN, 0.5% agar in EBN, 0.001% fried pigskin in EBN and 1% egg white in EBN. These detection levels are capable of effectively detecting the adulterants in commercial samples.     

A method to assemble DNA fragments mimicking junctions of transgenic elements: Application to the AquAdvantage salmon

The detection of transgenic animals in food and environmental samples will require the development of reliable screening assays. The identification of junction regions between genetic elements is the unequivocal way to prove the presence of transgene constructs. However, a sample from the transgenic organism or genetic construct is not always available for analysis. Here, we describe an easy-to-implement method to create junctions of genetic elements that can serve as DNA template or positive control for the development and validation of detection methods. The new methodology was successfully used to test several pairs of PCR primers intended for the detection of the first transgenic animal approved for human consumption (the AquAdvantage salmon). The junction region was created by the PCR amplification of a genetic element (antifreeze protein gene) with a tailed primer containing part of the adjacent element (growth hormone gene), yielding a PCR product with both regions fused. This strategy can be adapted to create junctions of genetic elements in any transgenic organism if reliable sequence data is available.     

Quantitative analysis of genetically modified organisms (GMO) in processed food by PCR-based methods

Two different PCR-based approaches for the quantitative analysis of genetically modified organism (GMO) – components in foods are presented using Soybean derived samples as an example. The first method – a double competitive PCR – is well suited to determine threshold levels of GMO content in food. The other – PCR on-line measurement – is suited to determine ratios of transgenic versus non-transgenic component. Both methods provide a means to alleviate the problems of standardisation encountered with simple qualitative PCR approaches and will allow to cope with threshold levels for GMO, once issued by legislative bodies.